This project will test the hypothesis that 5-hydroxytryptamine (5-HT, serotonin) and estrogen signaling interact to increase excitability of primary afferent neurons supplying the colon. Increased excitability is caused by changes in ion channel expression that result in visceral hypersensitivity in female rats. This hypothesis will be tested using male and female serotonin transporter (SERT) knockout (KO) rats, which we propose is a unique animal model of gender specific visceral hypersensitivity. The underlying pathophysiology of visceral pain is unclear and this is partly due to a lack of animal models where mechanistic and interventional studies can be conducted. However, published data indicate that alterations in 5-HT signaling may play a role in humans. In addition, visceral pain i more common in women than in men, suggesting that there are interactions between 5-HT and gender in the genesis of visceral pain. The overall hypothesis will be tested in 3 specific aims. Specific aim 1 will test the hypothesis that there is increased extracellular availability of 5-HT n vitro in the colon of wild type (WT) and SERT KO rats and that 5-HT release from enterochromaffin (EC) cells is unaffected by the SERT KO. Amperometry will measure 5-HT near the mucosa in response to mechanical and chemical mucosal stimulation. Immunohistochemical (IHC) and Western blot techniques will be used to verify SERT deletion and to assess 5-HT-containing EC cells in the gut of WT and SERT KO rats. IHC localization of the dopamine and norepinephrine transporters and organic cation transporters will be assessed to determine if their expression increases in SERT KO rats. Specific aim 2 will test the hypothesis that estrogen interacts with 5-HT to cause visceral hypersensitivity in female SERT KO rats. The visceromotor response to colorectal balloon distention will be used to measure visceral sensitivity in intact and ovariectomized female WT and SERT KO rats with and without estrogen replacement. In Specific Aim 3, the functional properties of colon projecting sensory neurons maintained in short term primary culture will be studied. These studies will test the hypothesis that colon projecting sensory nerves from female but not male SERT KO rats exhibit increased excitability when studied using whole cell patch clamp methods in vitro. The increased excitability is proposed to be due to interactions between 5-HT and estrogen on sensory neurons that lead to upregulation of tetrodotoxin-resistant sodium channels. These studies will show that increased 5-HT availability in female SERT KO rats alters visceral sensitivity as occurs in female human irritable bowel syndrome patients. The data would indicate that the SERT KO rat is a model for studying changes in the sensory nerve supply of the gut that leads to visceral hypersensitivity.